The present invention relates to a method of detecting DNA by DNA hybridization method with the use of fluorescent resonance energy transfer and reagents used therefor.
A DNA hybridization method has been widely utilized for diagnoses of genetic and infectious diseases. Most conventional methods for target DNA detection based on the DNA hybridization method are time-consuming and troublesome because they require many procedures such as immobilization of the target DNA on the solid support and washing of hybridized DNA duplex from the excess probe DNA. A method for detecting DNA based on fluorescence resonance energy transfer (FRET) has been proposed as a method to avoid these problems. That is, it is the method to detect DNA in a homogenous solution by preparing two nucleic acid probes (DNA probes) labeled with compounds to be an energy donor and an acceptor at the terminus and utilizing resonance energy transfer (sensitized luminescence of the acceptor) induced when these two nucleic acid probes (DNA probes) bind to the target DNA.
However, because organic dyes have been primarily utilized as the energy donor and the acceptor in conventional systems, a background luminescence (luminescence based on luminescence of the donor and the direct excitation of the acceptor) affecting a sensitized luminescence of the acceptor is significantly large resulting in no report of a practical system applicable for target DNA detection.
In the lights of the above circumstance, the objective of the present invention is to provide a practically usable method for detecting DNA by a DNA hybridization method with the use of fluorescent resonance energy transfer wherein the background luminescence (i.e., luminescence based on the luminescence of a donor and the direct excitation of an acceptor) affecting the sensitized luminescence of the acceptor is minimized so that a target DNA duplex can be conveniently and very easily detected in a homogenous solution.
The present invention relates to (1) a method for detecting DNA by the DNA hybridization method using as detection reagents, streptoavidin labeled with a fluorescent rare earth metal complex, a nucleic acid probe modified with biotin and a nucleic acid probe labeled with an organic cyanine dye, and taking advantage of fluorescent resonance energy transfer.
Also, the present invention relates to (2) a method for detecting DNA by the DNA hybridization method using a fluorescent rare earth metal complex as an energy donor, using an organic cyanine dye as an acceptor and taking advantage of fluorescent resonance energy transfer.
Further, the present invention relates to (3) a nucleic acid probe labeled with a fluorescent rare earth metal complex via biotin-streptoavidin.
Still, the present invention relates to a reagent kit comprising a nucleic acid probe modified with biotin, a nucleic acid probe modified with an organic cyanine dye and streptoavidin labeled with a fluorescent rare earth metal complex.
Still further, the present invention relates to (4) a reagent kit comprising a nucleic acid probe modified with biotin, a nucleic acid probe modified with a compound represented by the following chemical formula (II), and streptoavidin labeled with 4,4xe2x80x2-bis(1xe2x80x3,1xe2x80x3,1xe2x80x3,2xe2x80x3,2xe2x80x3,3xe2x80x3,3xe2x80x3-heptafluoro-4xe2x80x3,6xe2x80x3-hexanedione-6xe2x80x3-yl)-chlorosulfo-o-terphenyl (BHHCT)/Eu3+complex: 
(wherein MMTO represents 4-monomethoxytrityl group.)
Also, the present invention relates to (5) reagents for the method for detecting DNA by the DNA hybridization method taking advantage of fluorescent resonance energy transfer, comprising streptoavidin labeled with a fluorescent rare earth metal complex.
Further, the present invention relates to (6) a process for producing the nucleic acid probe described in the above (3), characterized in that the nucleic acid probe modified with biotin and the nucleic acid probe modified with the organic cyanine dye are mixed with a sample containing a target DNA, hybridized, and subsequently reacted with streptoavidin labeled with the fluorescent rare earth metal complex.
And also, the present invention relates to a method for detecting a target DNA by mixing a sample containing the DNA with a nucleic acid probe modified with biotin and a nucleic acid probe modified with an organic cyanine dye followed by hybridizing, then reacting streptoavidin labeled with a fluorescent rare earth metal complex therewith to further introduce the fluorescent rare earth metal complex, and subsequently measuring fluorescence of the DNA duplex.